1. Field of the Invention
The present invention relates to stable muteins of type 1 Placental Growth Factor (PLGF-1), their preparation, their therapeutic and cosmetic use and pharmaceutical and cosmetic compositions containing said derivatives. The invention likewise relates to the production of antibodies to said derivatives and their use in the diagnosis and treatment of tumoral and non-tumoral pathologies.
2. Description of Related Art
Type 1 Placental Growth Factor (PLGF-1) is an angiogenic homodimeric glycoprotein. Angiogenic activity relates to the dimeric form, as the monomeric form is inactive. The complete polynucleotide sequence encoding the PLGF-1 protein, along with its polypeptide sequence, were described by Maglione and Persico in Pat. EP-B-0 550 519 (WO-A-92/06194).
The above patent describes a method for producing PLGF-1 in bacteria modified using an inducible expression system, said method involving, after induction, bacterial lysis and direct extraction of the raw protein from the lysate solution. The protein obtained in this way shows low levels of biological activity.
A method for extraction and purification of the raw placental factor, obtained by expression in bacteria, is described by Maglione et. al. in patent application PCT/IT02/00065. The method involves a series of extraction, renaturation and purification passages, which as a whole make it possible to obtain the pure protein in an essentially dimeric form, that is to say in its most active form. It is, in fact, known that the monomeric form of the protein is biologically inactive, and only acquires angiogenic functions after renaturation to the dimeric form.
However, the present inventors have observed that the protein in dimeric form is partially unstable, and gives rise, in an aqueous solution, during storage or processing, to multimeric forms that show less biological activity and for this reason are less suitable for therapeutic use, due to the uncertainty of doses and biological activity.
The aim of the present invention is therefore to solve the problem of poor chemical/biological stability of PLGF-1 as observed mainly when the latter is conserved in aqueous solutions.